The DNA of a cell presents the first line of control over the metabolism and regulatory processes of the cell. However, the mechanisms by which the genetic message of a cell, residing in the DNA, is maintained and transmitted to offspring are unknown. In part our lack of knowledge regarding these processes is due to our failure to duplicate intracellular conditions in vitro. The proposed research will utilize a bacterial in vitro system which is capable of replicative DNA synthesis to investigate the mechanisms of DNA replication. Toluene- treated Escherichia coli cells offer a control over replicative synthesis not available in vivo. Efforts will be directed towards defining the enzymatic mechanisms required for the replicative process and the extent of each function. The possibility of overlapping or "back-up" activities of the several DNA polymerases will be investigated. The major control process of termination of replication will be studied using the in vitro technique to evaluate the mechanism of arrest of replication. Also the means of action of Bleomycin, an anti-tumor agent, will be studied. Studies with purified DNA polymerase II will be directed to defining its "editorial" function. There are numerous advantages to a prokaryote system at this stage of investigation of chromosomal duplication. General conclusions from this system will hopefully prove applicable to higher organisms. If the chromosome of the cell contains degenerate, uninterpretable, or deleterious information, then the functional capacity of the cell will be impaired. Such defects may be the basis of cancerous changes in mammalian cells, structural defects in higher organisms, or metabolic aberrations in either prokaryotes or eukaryotes. An understanding of the mechanisms of duplication of genetic information will be required to elucidate the basis of diseases in the categories noted.